The cytochrome P450 superfamily of enzymes exhibits an impressive range of chemical activities and biological roles. Nature has exploited these diverse enzymes for everything from steroid biosynthesis to interspecies chemical warfare, protection of the organisms from toxic compounds, drug detoxification and utilization of new food sources. (Ortiz de Montellano, P. R., Cytochrome P450: Structure, Mechanism, and Biochemistry (New York: Plenum Press) (1995); Gotoh, O., Cytochrome P450, 2nd Edition, pp. 255-272 (1993); Gonzalez et al., Trends Genet, 6:182-186 (1990); Mauersberger et al., Z Alig Mikrobiol., 121:313-321 (1981); Porter et al., J. Biol. Chem., 266:13469-13472 (1991)).
While activities are diverse across the entire superfamily, individual members of the superfamily show a much narrower range of catalytic activities, usually catalyzing oxygen insertion into C—H bonds and substrate specificities. The heme prosthetic group recruited by the cytochrome P450s to effect monooxygenation is also used by these and other proteins for oxygen transport, electron transfer, reduction, dealkylation and dehalogenation. (Sono et al., Chem Rev 96, 2841-2888 (1996), Eichhorn et al., Heme Proteins, New York, N.Y.: Elsevier Science Publishing Co., Inc. (1988)).
Recent engineering efforts have demonstrated that P450s can acquire new or improved activities by point mutation (Cirino, P. C., and Arnold, F. H., Angew Chem Int Ed Engl 42, 3299-3301 (2003); Li et al., Biochem Biophys. Acta., 1545:114-121 (2001); Glieder et al., Nat. Biotechnol. 20:1135-1139 (2002); Li et al., Chemistry, 6:1531-1536 (2000); Joo et al., Nature, 399:670-673 (1999); Appel et al., J. Biotechnol., 88:167-171 (2001)). However, making and characterizing each engineered P450 can be a laborious and time-consuming process. This is especially true when many of the mutations made, if done randomly, result in proteins with little or no function. This is even further exacerbated if one desires high levels of mutation. One possible way around this is to create libraries containing potentially numerous useful proteins by recombining different P450s either from nature or which have created in the laboratory. This could at least reduce the initial amount of work involved in creating the enzymes, although a substantial amount of work will still be required in testing these libraries.
However, creating protein libraries is not a simple matter, especially for P450s. P450s typically exhibit low sequence identity. Thus, annealing-based DNA -shuffling techniques are largely useless for creating highly diverse libraries of P450 chimeras or for creating the individual chimeric proteins (Stemmer, W P, Proc. Natl. Acad. Sci. USA, 91:10747-10751 (1994); Coco, W M. et al., Nat. Biotechnol., 19:354-359 (2001); Zhao, H. et al. (1998). Nat. Biotechnol., 16:258-261; Volkov, A A et al., Nucleic Acids Res., 27, e18 (1999); Kikuchi, M. et al., Gene, 243:133-137 (2000)). While there are some methods for making shuffled gene libraries independent of sequence homology, these approaches generate few crossovers and large numbers of inactive sequences due to insertions, deletions, and frameshifts, as well as disruptive crossover events. (Lutz, S. et al. Proc Natl Acad Sci USA, 98:11248-11253 (2001); Sieber, V. et al., Nat Biotechnol., 19:456-460 (2001)). Additionally, functional characterization of such libraries is extremely difficult without a selection method that can remove unfolded or nonfunctional sequences. Finally, as such libraries do not currently generally exist, the functional abilities, characteristics, benefits of, and method of characterizing such libraries currently remain unknown, although attempts have been made (Abecassis et al., Nucleic Acids Res 28:E88, (2000)).